Fibroblast growth factor 11

ABSTRACT

Disclosed is a human Fibroblast growth factor-11 polypeptide and DNA(RNA) encoding such polypeptide. Also provided is a procedure for producing such polypeptide by recombinant techniques. Also disclosed are methods for utilizing such polypeptide for promoting wound healing for example as a result of burns and ulcers, to prevent neuronal damage associated with stroke and due to neuronal disorders and promote neuronal growth, and to prevent skin aging and hair loss, to stimulate angiogenesis, mesodermal induction in early embryos and limb regeneration. Antagonists against such polypeptides and their use as a therapeutic to prevent abnormal cellular proliferation, hyper-vascular diseases and epithelial lens cell proliferation are also disclosed. Diagnostic methods for detecting mutations in the coding sequence and alterations in the concentration of the polypeptides in a sample derived from a host are also disclosed.

This application is a Divisional application entitled to prioritybenefits under 35 U.S.C. §120 for the information set forth in Ser. No.08/464,590, filed Jun. 5, 1995, issued as U.S. Pat. No. 5,763,214, onJun. 9, 1998.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention have been putatively identified as fibroblast growthfactor/heparin binding growth factor, hereinafter referred to as"FGF-11". The invention also relates to inhibiting the action of suchpolypeptides.

Fibroblast growth factors are a family of proteins characteristic ofbinding to heparin and are, therefore, also called heparin bindinggrowth factors (HBGF). Expression of different members of these proteinsare found in various tissues and are under particular temporal andspatial control. These proteins are potent mitogens for a variety ofcells of mesodermal, ectodermal, and endodermal origin, includingfibroblasts, corneal and vascular endothelial cells, granulocytes,adrenal cortical cells, chondrocytes, myoblasts, vascular smooth musclecells, lens epithelial cells, melanocytes, keratinocytes,oligodendrocytes, astrocytes, osteoblasts, and hematopoietic cells.

Each member has functions overlapping with others and also has itsunique spectrum of functions. In addition to the ability to stimulateproliferation of vascular endothelial cells, both FGF-1 and 2 arechemotactic for endothelial cells and FGF-2 has been shown to enableendothelial cells to penetrate the basement membrane. Consistent withthese properties, both FGF-1 and 2 have the capacity to stimulateangiogenesis. Another important feature of these growth factors is theirability to promote wound healing. Many other members of the FGF familyshare similar activities with FGF-1 and 2 such as promoting angiogenesisand wound healing. Several members of the FGF family have been shown toinduce mesoderm formation and to modulate differentiation of neuronalcells, adipocytes and skeletal muscle cells.

Other than these biological activities in normal tissues, FGF proteinshave been implicated in promoting tumorigenesis in carcinomas andsarcomas by promoting tumor vascularization and as transforming proteinswhen their expression is deregulated.

The FGF family presently consists of eight structurally-relatedpolypeptides: basic FGF, acidic FGF, int 2, hst 1/k-FGF, FGF-5, FGF-6,keratinocyte growth factor, AIGF (FGF-8) and recently a glia-activatingfactor has been shown to be a novel heparin-binding growth factor whichwas purified from the culture supernatant of a human glioma cell line(Miyamoto, M. et al., Mol. and Cell. Biol., 13(7):4251-4259 (1993). Thegenes for each have been cloned and sequenced. Two of the members, FGF-1and FGF-2, have been characterized under many names, but most often asacidic and basic fibroblast growth factor, respectively. The normal geneproducts influence the general proliferation capacity of the majority ofmesoderm and neuroectoderm-derived cells. They are capable of inducingangiogenesis in vivo and may play important roles in early development(Burgess, W. H. and Maciag, T., Annu. Rev. Biochem., 58:575-606,(1989)).

Many of the above-identified members of the FGF family also bind to thesame receptors and elicit a second message through binding to thesereceptors.

A eukaryotic expression vector encoding a secreted form of FGF-1 hasbeen introduced by gene transfer into porcine arteries. This modeldefines gene function in the arterial wall in vivo. FGF-1 expressioninduced intimal thickening in porcine arteries 21 days after genetransfer (Nabel, E. G., et al., Nature, 362:844-6 (1993)). It hasfurther been demonstrated that basic fibroblast growth factor mayregulate glioma growth and progression independent of its role in tumorangiogenesis and that basic fibroblast growth factor release orsecretion may be required for these actions (Morrison, R. S., et al., J.Neurosci. Res., 34:502-9 (1993)).

Fibroblast growth factors, such as basic FGF, have further beenimplicated in the growth of Kaposi's sarcoma cells in vitro (Huang, Y.Q., et al., J. Clin. Invest., 91:1191-7 (1993)). Also, the cDNA sequenceencoding human basic fibroblast growth factor has been cloned downstreamof a transcription promoter recognized by the bacteriophage T7 RNApolymerase. Basic fibroblast growth factors so obtained have been shownto have biological activity indistinguishable from human placentalfibroblast growth factor in mitogenicity, synthesis of plasminogenactivator and angiogenesis assays (Squires, C. H., et al., J. Biol.Chem., 263:16297-302 (1988)).

U.S. Pat. No. 5,155,214 discloses substantially pure mammalian basicfibroblast growth factors and their production. The amino acid sequencesof bovine and human basic fibroblast growth factor are disclosed, aswell as the DNA sequence encoding the polypeptide of the bovine species.

Newly discovered FGF-9 has around 30% sequence similarity to othermembers of the FGF family. Two cysteine residues and other consensussequences in family members were also well conserved in the FGF-9sequence. FGF-9 was found to have no typical signal sequence in its Nterminus like those in acidic and basic FGF. However, FGF-9 was found tobe secreted from cells after synthesis despite its lack of a typicalsignal sequence FGF (Miyamoto, M. et al., Mol. and Cell. Biol.,13(7):4251-4259 (1993). Further, FGF-9 was found to stimulate the cellgrowth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3,and PC-12 cells but not that of human umbilical vein endothelial cells(Naruo, K., et al., J. Biol. Chem., 268:2857-2864 (1993).

Basic FGF and acidic FGF are potent modulators of cell proliferation,cell motility, differentiation, and survival and act on cell types fromectoderm, mesoderm and endoderm. These two FGFs, along with KGF andAIGF, were identified by protein purification. However, the other fourmembers were isolated as oncogenes., expression of which was restrictedto embryogenesis and certian types of cancers. FGF-9 was demonstrated tobe a mitogen against glial cells. Members of the FGF family are reportedto have oncogenic potency. FGF-9 has shown transforming potency whentransformed into BALB/c3T3 cells (Miyamoto, M., et al., Mol. Cell.Biol., 13(7):4251-4259 (1993).

Androgen induced growth factor (AIGF), also known as FGF-8, was purifiedfrom a conditioned medium of mouse mammary carcinoma cells (SC-3)simulated with testosterone. AIGF is a distinctive FGF-like growthfactor, having a putative signal peptide and sharing 30-40% homologywith known members of the FGF family. Mammalian cells transformed withAIGF shows a remarkable stimulatory effect on the growth of SC-3 cellsin the absence of androgen. Therefore, AIGF mediates androgen-inducedgrowth of SC-3 cells, and perhaps other cells, since it is secreted bythe tumor cells themselves.

The polypeptide of the present invention has been putatively identifiedas a member of the FGF family as a result of amino acid sequencehomology with other members of the FGF family.

In accordance with one aspect of the present invention, there areprovided novel mature polypeptides as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptides of the present invention are ofhuman origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding the polypeptides ofthe present invention, including mRNAs, DNAs, cDNAs, genomic DNA, aswell as antisense analogs thereof and biologically active anddiagnostically or therapeutically useful fragments thereof.

In accordance with still another aspect of the present invention, thereare provided processes for producing such polypeptides by recombinanttechniques through the use of recombinant vectors, such as cloning andexpression plasmids useful as reagents in the recombinant production ofthe polypeptides of the present invention, as well as recombinantprokaryotic and/or eukaryotic host cells comprising a nucleic acidsequence encoding a polypeptide of the present invention.

In accordance with a further aspect of the present invention, there isprovided a process for utilizing such polypeptides, or polynucleotidesencoding such polypeptides, for screening for agonists and antagoniststhereto and for therapeutic purposes, for example, promoting woundhealing for example as a result of burns and ulcers, to prevent neuronaldamage associated with stroke and due to neuronal disorders and promoteneuronal growth, and to prevent skin aging and hair loss, to stimulateangiogenesis, mesodermal induction in early embryos and limbregeneration.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists against such polypeptides and processes fortheir use to inhibit the action of such polypeptides, for example, inthe treatment of cellular transformation, for example, tumors, to reducescarring and treat hyper-vascular diseases.

In accordance with another aspect of the present invention, there areprovided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to a polynucleotide encodinga polypeptide of the present invention.

In accordance with yet another aspect of the present invention, thereare provided diagnostic assays for detecting diseases or susceptibilityto diseases related to mutations in a nucleic acid sequence of thepresent invention and for detecting over-expression of the polypeptidesencoded by such sequences.

In accordance with another aspect of the present invention, there isprovided a process for utilizing such polypeptides, or polynucleotidesencoding such polypeptides, for in vitro purposes related to scientificresearch, synthesis of DNA and manufacture of DNA vectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are meant only as illustrations of specificembodiments of the present invention and are not meant as limitations inany manner.

FIGS. 1A and 1B collectively show the polynucleotide sequence (SEQ IDNO:1) of the cDNA encoding the mature human FGF-11 polypeptide with itsdeduced amino acid sequence (SEQ ID NO:2), wherein FIG. 1A illustratesthe first portions of the polynucleotide sequence of the cDNA encodingthe mature human FGF-11 polypeptide with its deduced amino acid sequenceand FIG. 1B continues consecutively with the latter portions,respectively, up to and including the end of the same polynucleotide andamino acid sequence. The standard one-letter abbreviations for aminoacid residues are used to illustrate the amino acid sequence in FIGS. 1Aand 1B.

FIGS. 2A, 2B and 2C collectively show polypeptide sequences in alignmentand consecutively present the alignment of the sequences to illustratean amino acid sequence comparison between FGF-11 according to thepresent invention (second line of each comparative row; SEQ ID NO:2) andeach of the following FGF polypeptide sequences, respectively: FGF-10(first line of each comparative row, SEQ ID NO:9); FGF-9 (third line,SEQ ID NO:10); FGF-3 (fourth line, SEQ ID NO:11); FGF-7 (fifth line, SEQID NO:12); FGF-1 (sixth line, SEQ ID NO:13); FGF-2 (seventh line, SEQ IDNO:4), FGF-4 (eighth line, SEQ ID NO:15); FGF-6 (ninth line, SEQ IDNO:16) and FGF-5 (tenth line, SEQ ID NO:17). One-letter abbreviationsare utilized for the amino acid residues in FIGS. 2A-2C.

In accordance with one aspect of the present invention, there areprovided isolated nucleic acids molecules (polynucleotides) which encodefor the mature polypeptide having the deduced amino acid sequence ofFIGS. 1A-1B collectively (SEQ ID NOS:2) or for the mature polypeptideencoded by the cDNA of the clone deposited as ATCC Deposit No. 97150 onMay 12, 1995 as a biological deposit with the ATCC, 10801 UniversityBoulevard, Manassas Va. 20110-2209.

The polynucleotide encoding FGF-11 of this invention was discoveredinitially in a cDNA library derived from 9 week old early stage humantissue. The FGF-11 polypeptide is structurally related to all members ofthe fibroblast growth factor family and contains an open reading frameencoding a polypeptide of 255 amino acids. Among the top matches are: 1)42% identity and 65% sequence similarity to FGF-9 over a stretch of 127amino acids; 2) 37% identity and 64% similarity with FGF-7 (keratinocytegrowth factor) in a region of 87 amino acids; 3) 38% identity and 64%similarity with FGF-1 (acidic FGF) over a stretch of 120 amino acids.

The FGF/HBGF family signature, GXLX(S,T,A,G)X6(D,E)CXFXE is conserved inthe polypeptide of the present invention, (X means any amino acidresidue; (D,E) means either D or E residue; X6 means any 6 amino acidresidues).

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded. The codingsequence which encodes the mature polypeptide may be identical to thecoding sequence shown in FIGS. 1A-1B collectively (SEQ ID NOS:1) or thatof the deposited clone or may be a different coding sequence, as aresult of the redundancy or degeneracy of the genetic code, encodes thesame, mature polypeptide as the DNA of FIGS. 1A-1B, collectively, (SEQID NOS:1) or the deposited cDNA.

The polynucleotides which encodes for the mature polypeptide of FIGS.1A-1B collectively (SEQ ID NOS:2) or for the mature polypeptides encodedby the deposited cDNA(s) may include: only the coding sequence for themature polypeptide; the coding sequence for the mature polypeptide andadditional coding sequence such as a leader or secretory sequence or aproprotein sequence; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5' and/or 3' of the coding sequence forthe mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptides having the deduced amino acid sequenceof FIGS. 1A-1B collectively (SEQ ID NOS:2) or the polypeptides encodedby the cDNA(s) of the deposited clone(s). The variants of thepolynucleotide may be a naturally occurring allelic variant of thepolynucleotide or a non-naturally occurring variant of thepolynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIGS. 1A-1B collectively (SEQ ID NOS:2)or the same mature polypeptides encoded by the cDNA(s) of the depositedclone (s) as well as variants of such polynucleotides which variantsencode for a fragment, derivative or analog of the polypeptide of FIGS.1A-1B collectively (SEQ ID NOS:2) or the polypeptides encoded by thecDNA(s) of the deposited clone(s). Such nucleotide variants includedeletion variants, substitution variants and addition or insertionvariants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 1A-1B, collectively (SEQ ID NOS:1) or of the codingsequence of the deposited clone(s). As known in the art, an allelicvariant is an alternate form of a polynucleotide sequence which may havea substitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptides.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptides may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5' amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotides of the present invention mayencode for a mature protein, or for a protein having a prosequence orfor a protein having both a prosequence and a presequence (leadersequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term "gene" means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length FGF-11 gene may be used as a hybridizationprobe for a cDNA library to isolate the full length gene and to isolateother genes which have a high sequence similarity to the gene or similarbiological activity. Probes of this type preferably have at least 30bases and may contain, for example, 50 or more bases. The probe may alsobe used to identify a cDNA clone corresponding to a full lengthtranscript and a genomic clone or clones that contain the completeFGF-11 gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe FGF-11 gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term"stringent conditions" means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIGS. 1A-1B collectively(SEQ ID NO:1) or the deposited cDNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferablyat least 30 bases, and more preferably at least 50 bases which hybridizeto a polynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the purposes of Patent Procedure. These deposits are provided merelyas a convenience and are not an admission that a deposit is requiredunder 35 U.S.C. § 112. The sequence of the polynucleotides contained inthe deposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with the description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

The present invention further relates to an FGF polypeptide which hasthe deduced amino acid sequence of FIGS. 1A-1B, collectively (SEQ IDNOS:2) or which has the amino acid sequence encoded by the depositedcDNA(s), as well as fragments, analogs and derivatives of suchpolypeptides.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIGS. 1A-1B collectively (SEQ ID NOS:2) or those encodedby the deposited cDNA(s), means polypeptides which retains essentiallythe same biological function or activity as such polypeptides. Thus, ananalog includes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides or synthetic polypeptides, preferablyrecombinant polypeptides.

The fragment, derivative or analog of the polypeptide of FIGS. 1A-1Bcollectively (SEQ ID NOS:2) or that encoded by the deposited cDNA(s) maybe (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofSEQ ID NO:2 and still more preferably at least 95% similarity (stillmore preferably at least 95% identity) to the polypeptide of SEQ ID NO:2and also include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells may be genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the FGF genes. The culture conditions, suchas temperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The polynucleotide of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, for example,the polynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,and pseudorabies. However, any other vector or plasmid may be used aslong as they are replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease sites by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain a gene to providea phenotypic trait for selection of transformed host cells such asdihydrofolate reductase or neomycin resistance for eukaryotic cellculture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as herein abovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned; bacterial cells, such as E. coli, Salmonellatyphimurium, Streltogmces; fungal cells, such as yeast; insect cells,such as Drosophila S2 and Spodoptera Sf9; animal cells such as CEO, COSor Bowes melanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a,pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described construct. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, 1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), thedisclosure of which is hereby incorporated by reference.

Transcription of a DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication origin,and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation, initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation, initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,U.S.A.). These pBR322 "backbone" sections are combined with anappropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector; for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 viralgenome, for example, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

The polypeptide of the present invention may be recovered-and purifiedfrom recombinant cell cultures by methods used heretofore, includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxyapatite chromatography and lectin chromatography. Proteinrefolding steps can be used, as necessary, in completing configurationof the mature protein. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps.

The polypeptide of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated with mammalian or other eukaryotic carbohydrates or may benon-glycosylated. Polypeptides of the invention may also include aninitial methionine amino acid residue.

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration.

The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat and prevent neuronal damage associated withstroke and which occurs in certain neuronal disorders orneuro-degenerative conditions such as Alzheimer's disease, Parkinson'sdisease, and AIDS-related complex. FGF-11 as the ability to stimulatechondrocyte growth, therefore, they may be employed to enhance bone andperiodontal regeneration and aid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The FGF-11 polypeptide may also be employed for preventing hair loss,since FGF family members activate hair-forming cells and promotesmelanocyte growth. Along the same lines, the polypeptides of the presentinvention may be employed to stimulate growth and differentiation ofhematopoietic cells and bone marrow cells when used in combination withother cytokines.

The FGF-11 polypeptide may also be employed to maintain organs beforetransplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA, manufacture of DNAvectors and for the purpose of providing diagnostics and therapeuticsfor the treatment of human disease.

This invention provides a method for identification of the receptors forthe polypeptides of the present invention. The genes encoding thereceptor can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting (Coligan, etal., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the the polypeptide of the presentinvention, after they have been labelled. The polypeptides can belabeled by a variety of means including iodination or inclusion of arecognition site for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

This invention provides a method of screening compounds to identifythose which modulate the action of the polypeptide of the presentinvention. An example of such an assay comprises combining a mammalianfibroblast cell, a the polypeptide of the present invention, thecompound to be screened and ³ [H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of ³ [H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of ³ [H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the FGF-11 receptor is measured and the ability of thecompound to bind to the receptor and elicit a second messenger responseis measured to determine if the compound is a potential agonist orantagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, tyrosine phosphorylation, ion channels orphosphoinositide hydrolysis.

Examples of antagonist compounds include antibodies, or in some cases,oligonucleotides, which bind to the receptor for the polypeptide of thepresent invention but elicit no second messenger response or bind to theFGF-11 polypeptide itself. Alternatively, a potential antagonist may bea mutant form of the polypeptide which binds to the receptors, however,no second messenger response is elicited and, therefore, the action ofthe polypeptide is effectively blocked.

Another antagonist compound to the FGF-11 gene and gene product is anantisense construct prepared using antisense technology. Antisensetechnology can be used to control gene expression through triple-helixformation or antisense DNA or RNA, both of which methods are based onbinding of a polynucleotide to DNA or RNA. For example, the 5' codingportion of the polynucleotide sequence, which encodes for the maturepolypeptides of the present invention, is used to design an antisenseRNA oligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix--see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of the polypeptides of the present invention. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the polypeptide (Antisense--Okano,J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the polypeptide.

Potential antagonist compounds also include small molecules which bindto and occupy the binding site of the receptors thereby making thereceptor inaccessible to its polypeptide such that normal biologicalactivity is prevented. Examples of small molecules include, but are notlimited to, small peptides or peptide-like molecules.

Antagonist compounds may be employed to inhibit the cell growth andproliferation effects of the polypeptides of the present invention onneoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonists may also be employed to prevent hyper-vascular diseases,and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonists may also be employed to prevent the growth of scartissue during wound healing.

The antagonists may be employed in a composition with a pharmaceuticallyacceptable carrier, e.g., as hereinafter described.

The polypeptides, agonists and antagonists of the present invention maybe employed in combination with a suitable pharmaceutical carrier tocomprise a pharmaceutical composition for parenteral administration.Such compositions comprise a therapeutically effective amount of thepolypeptide, agonist or antagonist and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides, agonists and antagonists of the present invention may beemployed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmaimer such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10 μg/kgbody weight and in most cases they will be administered in an amount notin excess of about 8 mg/Kg body weight per day. In most cases, thedosage is from about 10 μg/kg to about 1 mg/kg body weight daily, takinginto account the routes of administration, symptoms, etc. In thespecific case of topical administration, dosages are preferablyadministered from about 0.1 μg to 9 mg per cm².

The polypeptide of the invention and agonist and antagonist compoundswhich are polypeptides, may also be employed in accordance with thepresent invention by expression of such polypeptide in vivo, which isoften referred to as "gene therapy."

Thus, for example, cells may be engineered with a polynucleotide (DNA orRNA) encoding for the polypeptide ex vivo, the engineered cells are thenprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, cells may be engineered byprocedures known in the art by use of a retroviral particle containingRNA encoding for the polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of thepolypeptide in vivo, for example, by procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such methods should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retroviral particle, for example, an adenovirus, which may beused to engineer cells in vivo after combination with a suitabledelivery vehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CTV) promoter described inMiller, et al., Biotechnipues, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CKV) promoter; therespiratory syncytial-virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PES501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence (s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequencers) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the genes of the presentinvention as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutations in thenucleic acid sequences encoding the polypeptide of the presentinvention.

Individuals carrying mutations in a gene of the present invention may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding a polypeptide of thepresent invention can be used to identify and analyze mutations. Forexample, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to radiolabeledRNA or alternatively, radiolabeled antisense DNA sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNaseA digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, U.S.A., 85:4397-4401(1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of FGF-11 proteins in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of abnormal cellular proliferation, forexample, a tumor. Assays used to detect levels of protein in a samplederived from a host are well-known to those of skill in the art andinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis, ELISA assays and "sandwich" assay. An ELISA assay (Coligan, etal., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initiallycomprises preparing an antibody specific to an antigen to thepolypeptides of the present invention, preferably a monoclonal antibody.In addition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or, in this example, a horseradishperoxidase enzyme. A sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein like bovine serum albumen. Next,the monoclonal antibody is incubated in the dish during which time themonoclonal antibodies attach to any polypeptides of the presentinvention attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is now placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to the protein ofinterest.

Unattached reporter antibody is then washed out. Peroxidase substratesare then added to the dish and the amount of color developed in a giventime period is a measurement of the amount of a polypeptide of thepresent invention present in a given volume of patient sample whencompared against a standard curve.

A competition assay may be employed wherein antibodies specific to apolypeptide of the present invention are attached to a solid support andlabeled FGF-11 and a sample derived from the host are passed over thesolid support and the amount of label detected, for example by liquidscintillation chromatography, can be correlated to a quantity of apolypeptide of the present invention in the sample.

A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay apolypeptide of the present invention is passed over a solid support andbinds to antibody attached to a solid support. A second antibody is thenbound to the polypeptide of interest. A third antibody which is labeledand specific to the second antibody is then passed over the solidsupport and binds to the second antibody and an amount can then bequantified.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphism's) are presently available for markingchromosomal location. The mapping of DHAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3'untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 50 or 60bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man (available on line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and diseases that have been mapped to the same chromosomal regionare then identified through linkage analysis (coinheritance ofphysically adjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples,certain-frequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by t he manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase ("ligase")per 0.5 μg of approximately equiuolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described bythe method of Graham, P. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Purification of FGF-11 Proteins

The DNA sequence encoding FGF-11, ATCC # 97150, is initially amplifiedusing PCR oligonucleotide primers corresponding to the 5' sequences ofthe processed protein (minus the signal peptide sequence) and the vectorsequences 3' to the gene. Additional nucleotides corresponding to thegene are added to the 5' and 3' sequences respectively. The 5'oligonucleotide primer 5' CGCGGATCCATCATGAGTGGAAAGGTGACCAAG 3' (SEQ IDNO:3) contains a BamHI restriction enzyme site. The 3' sequence 5'CGCGGATCCCGTTGATTCATTGTGGCTCAT 3' (SEQ ID NO:4) contains complementarysequences to a BamHI site and is followed by 21 nucleotides of FGF-11coding sequence.

The restriction enzyme sites correspond to the restriction enzyme siteson the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth,Calif. 91311). pQE-60 encodes antibiotic resistance (Ampr), a bacterialorigin of replication (ori), an IPTG-regulatable promoter operator(P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzymesites. pQE-60 was then digested with NcoI and BamHI. The amplifiedsequences are ligated into pQE-60 and are inserted in frame with thesequence encoding for the histidine tag and the ribosome binding site(RBS). The ligation mixture is then used to transform E. coli strainM15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. etal., Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989). M15/rep4 contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies were selected.Plasmid DNA is isolated and confirmed by restriction analysis. Clonescontaining the desired constructs are grown overnight (O/N) in liquidculturein LB media supplemented with both Amp (100 μg/ml) and Kan (25μg/ml). The O/N culture is used to inoculate a large culture at a ratioof 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalactopyranoside") is then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells a re grown an extra 3 to 4 hours. Cellsare then harvested by centrifugation. The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized FGF-11 is purified from this solution by chromatography on aNickel-NAT resin under conditions that allow for tight binding byproteins containing the 5-His tag (Hochuli, E. et al.S , J.Chromatography 411:177-184 (1984)) . The proteins are eluted from thecolumn in 6 molar guanidine HCl pH 5.0 and for the purpose ofrenaturation adjusted to 3 molar guanidine HCl, 100 mM sodium phosphate,10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized).After incubation in this solution for 12 hours the proteins are dialyzedto 10 mmolar sodium phosphate.

EXAMPLE 2 Expression of FGF-11 by in vitro Transcription andTranslation.

The FGF-11 cDNA, ATCC # 97150, was transcribed and translated in vitroto determine the size of the translatable polypeptide encoded by thefull length FGF-11 cDNA. The full length cDNA inserts of FGF-11 in thepbluescript SK vector.

The in vitro transcription/translation reaction was performed in a 25 ulvolume, using the T_(N) T™ Coupled Reticulocylae Lysate Systems(Promega, CAT# L4950). Specifically, the reaction contains 12.5 ul ofT_(N) T rabbit reticulocyte lysate, 2 μl of T_(N) T reaction buffer, 1μl of T3 polymerase, 1 μl of 1 mM amino acid mixture (minus methionine),4 μl of ³⁵ S-methionine (>1000 Ci/mmol, 10 mCi/ml), 1 μl of 40 U/μl;RNasin ribonuclease inhibitor, 0.5 or 1 μg of pBluescript FGF-11plasmid. Nuclease-free H₂ O was added to bring the volume to 25 ul. Thereaction was incubated at 30° C. for 2 hours . Five microliters of thereaction product was analyzed on a 4-20% gradient SDS-PAGE gel. Afterfixing in 25% isopropanol and 10% acetic acid, the gel was dried andexposed to an X-ray film overnight at 70° C.

EXAMPLE 3 Cloning and Expression of FGF-11 Using the BaculovirusExpression System

The DNA sequence encoding the full length FGF-11 protein, ATCC # 97150,is amplified using PCR oligonucleotide primers corresponding to the 5'and 3' sequences of the gene:

The FGF-11 5' primer has the sequence 5' CGCGGATCCATCATGAGTGGAAAGGTGACCAAG 3' (SEQ ID NO:5) and contains a BamHI restrictionenzyme site (in bold) such that cloning at this site will put thebaculovirus signal sequence in frame with 21 nucleotides of the FGF-11gene downstream of the putative FGF-11 signal peptide cleavage site.

The 3' primer has the sequence 5' CGCGGTACCCTACGTTGA TTCATTGTGGCT 3'(SEQ ID NO:6) and contains the cleavage site for the restrictionendonuclease Asp718 and 21 nucleotides complementary to the 3'non-translated sequence of the gene.

The amplified sequences are isolated from a 1% agarose gel using acommercially available kit ("Geneclean," BIO 101 Inc., La Jolla,Calif.). The fragment is then digested with the respective endonucleasesand purified again on a 1% agarose gel. This fragment is designated F2.

The vector pA2 (modification of pVL941 vector, discussed below) is usedfor the expression of the proteins using the baculovirus expressionsystem (for review see: Summers, M.D. and Smith, G.E. 1987, A manual ofmethods for baculovirus vectors and insect cell culture procedures,Texas Agricultural Experimental Station Bulletin No. 1555). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI andAsp718. The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E.coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pA2 such as pRG1, pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes anddephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA is then isolated from a 1% agarose gel using thecommercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E.coli DH5α cells are then transformed and bacteria identifiedthat contained the plasmid (pBacFGF-11) using the respective restrictionenzymes. The sequence of the cloned fragment are confirmed by DNAsequencing.

5 μg of the plasmid pBacFGF-11 are co-transtected with 1.0 μg of acommercially available linearized baculovirus ("BaculoGold™ baculovirusDNA", Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. U.S.A., 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmids, in each case,are mixed in a sterile well of microtiter plates containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in 35 mm tissue culture plates with 1 ml Grace's medium withoutserum. The plates are rocked back and forth to mix the newly addedsolution. The plates are then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plates are put back into an incubator and cultivation continued at27° C. for four days.

After four days the supernatant is collected and plaque assays performedsimilar as described by Summers and Smith (supra). As a modification anagarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) isused which allows an easy isolation of blue stained plaques. (A detaileddescription of a "plaque assay" can also be found in the user's guidefor insect cell culture and baculovirology distributed by LifeTechnologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution the virus is added to the cells andblue stained plaques are picked with the tip of an Eppendorf pipette.The agar containing the recombinant viruses is then resuspended in anEppendorf tube containing 200 μl of Grace's medium. The agar is removedby a brief centrifugation and the supernatant containing the recombinantbaculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the supernatants of these culture dishes are harvested andthen stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-FGF-11 at a multiplicity of infection (MOI) of 2. Sixhours later the medium is removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵ S-methionine and 5 μCi ³⁵ S cysteine (Amersham)are added. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 4 Expression of Recombinant FGF-11 in COS Cells

The expression of plasmids, FGF-11-HA derived from a vector pcDNA3/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CKV promoter followedby a polylinker region, an SV40 intron and polyadenylation site. DNAfragments encoding the entire FGF-11 precursor and an HA tag fused inframe to the 3' end is cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CKVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein as previously described (I. Wilson, H.Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell37:767, (1984)). The infusion of HA tag to the target protein allowseasy detection of the recombinant protein with an antibody thatrecognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding FGF-11, ATCC # 97150, is constructed by PCRusing two primers: the 5' primer 5' CGCGGATCCATCATGAGTGGAAAGGTGACCAAG 3'(SEQ ID NO:7) contains a BamHI site followed by 21 nucleotides of codingsequence starting from the initiation codon; the 3' sequence 5' CTCGAGCGTTGATTCATTGTGGCTCAT 3' (SEQ ID NO:8) contains complementary sequencesto an XbaI site and the last 21 nucleotides of the FGF-11 codingsequence (not including the stop codon). Therefore, the PCR productcontains a XhoI site, coding sequence followed by HA tag fused in frame,a translation termination stop codon next to the HA tag, and an XhoIsite.

The PCR amplified DNA fragments and the vector, pcDNA3/Amp, are digestedwith the respective restriction enzymes and ligated. The ligationmfixture is transformed into E. coli strain SURE (available fromStratagene Cloning Systems, La Jolla, Calif. 92037) the transformedculture is plated on ampicillin media plates and resistant colonies areseleted. Plasstid DNA is isolated from transformants and examined byrestriction analysis for the presence of the correct fragment. Forexpression of the recombinant FGF-11 COS cells are transfected with theexpression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T.Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989)). The expression of the FGF-11-HA protein is detected byradiolabelling and immunoprecipitation method (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells are labelled for 8 hours with ³⁵ S-cysteine two days posttransfection. Culture media is then collected and cells are lysed withdetergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984) Both celllysate and culture media are precipitated with a n HA specificmonoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGEgels.

EXAMPLE 5 Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemerature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5' and 3' end sequencesrespectively. The 5' primer containing an EcoRI site and the 3' primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbeccol's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging ceils arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 17                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 768 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..765                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG AGT GGA AAG GTG ACC AAG CCC AAA GAG GA - #G AAA GAT GCT TCT AAG           48                                                                       Met Ser Gly Lys Val Thr Lys Pro Lys Glu Gl - #u Lys Asp Ala Ser Lys             1               5 - #                 10 - #                 15              - - GTT CTG GAT GAC GCC CCC CCT GGC ACA CAG GA - #A TAC ATT ATG TTA CGA           96                                                                       Val Leu Asp Asp Ala Pro Pro Gly Thr Gln Gl - #u Tyr Ile Met Leu Arg                        20     - #             25     - #             30                  - - CAA GAT TCC ATC CAA TCT GCG GAA TTA AAG AA - #A AAA GAG TCC CCC TTT          144                                                                       Gln Asp Ser Ile Gln Ser Ala Glu Leu Lys Ly - #s Lys Glu Ser Pro Phe                    35         - #         40         - #         45                      - - CGT GCT AAG TGT CAC GAA ATC TTC TGC TGC CC - #G CTG AAG CAA GTA CAC          192                                                                       Arg Ala Lys Cys His Glu Ile Phe Cys Cys Pr - #o Leu Lys Gln Val His                50             - #     55             - #     60                          - - CAC AAA GAG AAC ACA GAG CCG GAA GAG CCT CA - #G CTT AAG GGT ATA GTT          240                                                                       His Lys Glu Asn Thr Glu Pro Glu Glu Pro Gl - #n Leu Lys Gly Ile Val            65                 - # 70                 - # 75                 - # 80       - - ACC AAG CTA TAC AGC CGA CAA GGC TAC CAC TT - #G CAG CTG CAG GCG GAT          288                                                                       Thr Lys Leu Tyr Ser Arg Gln Gly Tyr His Le - #u Gln Leu Gln Ala Asp                            85 - #                 90 - #                 95              - - GGA ACC ATT GAT GGC ACC AAA GAT GAG GAC AG - #C ACT TAC ACT CTG TTT          336                                                                       Gly Thr Ile Asp Gly Thr Lys Asp Glu Asp Se - #r Thr Tyr Thr Leu Phe                       100      - #           105      - #           110                  - - AAC CTC ATC CCT GTG GGT CTG CGA GTG GTG GC - #T ATC CAA GGA GTT CAA          384                                                                       Asn Leu Ile Pro Val Gly Leu Arg Val Val Al - #a Ile Gln Gly Val Gln                   115          - #       120          - #       125                      - - ACC AAG CTG TAC TTG GCA ATG AAC AGT GAG GG - #A TAC TTG TAC ACC TCG          432                                                                       Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu Gl - #y Tyr Leu Tyr Thr Ser               130              - #   135              - #   140                          - - GAA CTT TTC ACA CCT GAG TGC AAA TTC AAA GA - #A TCA GTG TTT GAA AAT          480                                                                       Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys Gl - #u Ser Val Phe Glu Asn           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - TAT TAT GTG ACA TAT TCA TCA ATG ATA TAC CG - #T CAG CAG CAG TCA        GGC      528                                                                    Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr Ar - #g Gln Gln Gln Ser Gly                          165  - #               170  - #               175              - - CGA GGG TGG TAT CTG GGT CTG AAC AAA GAA GG - #A GAG ATC ATG AAA GGC          576                                                                       Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu Gl - #y Glu Ile Met Lys Gly                       180      - #           185      - #           190                  - - AAC CAT GTG AAG AAG AAC AAG CCT GCA GCT CA - #T TTT CTG CCT AAA CCA          624                                                                       Asn His Val Lys Lys Asn Lys Pro Ala Ala Hi - #s Phe Leu Pro Lys Pro                   195          - #       200          - #       205                      - - CTG AAA GTG GCC ATG TAC AAG GAG CCA TCA CT - #G CAC GAT CTC ACG GAG          672                                                                       Leu Lys Val Ala Met Tyr Lys Glu Pro Ser Le - #u His Asp Leu Thr Glu               210              - #   215              - #   220                          - - TTC TCC CGA TCT GGA AGC GGG ACC CCA ACC AA - #G AGC AGA AGT GTC TCT          720                                                                       Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr Ly - #s Ser Arg Ser Val Ser           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - GGC GTG CTG AAC GGA GGC AAA TCC ATG AGC CA - #C AAT GAA TCA ACG              76 - #5                                                                  Gly Val Leu Asn Gly Gly Lys Ser Met Ser Hi - #s Asn Glu Ser Thr                               245  - #               250  - #               255              - - TAG                  - #                  - #                  - #                768                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 255 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ser Gly Lys Val Thr Lys Pro Lys Glu Gl - #u Lys Asp Ala Ser Lys        1               5 - #                 10 - #                 15              - - Val Leu Asp Asp Ala Pro Pro Gly Thr Gln Gl - #u Tyr Ile Met Leu Arg                   20     - #             25     - #             30                  - - Gln Asp Ser Ile Gln Ser Ala Glu Leu Lys Ly - #s Lys Glu Ser Pro Phe               35         - #         40         - #         45                      - - Arg Ala Lys Cys His Glu Ile Phe Cys Cys Pr - #o Leu Lys Gln Val His           50             - #     55             - #     60                          - - His Lys Glu Asn Thr Glu Pro Glu Glu Pro Gl - #n Leu Lys Gly Ile Val       65                 - # 70                 - # 75                 - # 80       - - Thr Lys Leu Tyr Ser Arg Gln Gly Tyr His Le - #u Gln Leu Gln Ala Asp                       85 - #                 90 - #                 95              - - Gly Thr Ile Asp Gly Thr Lys Asp Glu Asp Se - #r Thr Tyr Thr Leu Phe                  100      - #           105      - #           110                  - - Asn Leu Ile Pro Val Gly Leu Arg Val Val Al - #a Ile Gln Gly Val Gln              115          - #       120          - #       125                      - - Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu Gl - #y Tyr Leu Tyr Thr Ser          130              - #   135              - #   140                          - - Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys Gl - #u Ser Val Phe Glu Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr Ar - #g Gln Gln Gln Ser        Gly                                                                                             165  - #               170  - #               175             - - Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu Gl - #y Glu Ile Met Lys Gly                  180      - #           185      - #           190                  - - Asn His Val Lys Lys Asn Lys Pro Ala Ala Hi - #s Phe Leu Pro Lys Pro              195          - #       200          - #       205                      - - Leu Lys Val Ala Met Tyr Lys Glu Pro Ser Le - #u His Asp Leu Thr Glu          210              - #   215              - #   220                          - - Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr Ly - #s Ser Arg Ser Val Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gly Val Leu Asn Gly Gly Lys Ser Met Ser Hi - #s Asn Glu Ser Thr                         245  - #               250  - #               255              - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CGCGGATCCA TCATGAGTGG AAAGGTGACC AAG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CGCGGATCCC GTTGATTCAT TGTGGCTCAT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CGCGGATCCA TCATGAGTGG AAAGGTGACC AAG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - CGCGGTACCC TACGTTGATT CATTGTGGCT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CGCGGATCCA TCATGAGTGG AAAGGTGACC AAG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - CTCGAGCGTT GATTCATTGT GGCTCAT          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 181 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Met Glu Ser Lys Glu Pro Gln Leu Lys Gly Il - #e Val Thr Arg Leu Phe      1               5   - #                10  - #                15               - - Ser Gln Gln Gly Tyr Phe Leu Gln Met His Pr - #o Asp Gly Thr Ile Asp                  20      - #            25      - #            30                   - - Gly Thr Lys Asp Glu Asn Ser Asp Tyr Thr Le - #u Phe Asn Leu Ile Pro              35          - #        40          - #        45                       - - Val Gly Leu Arg Val Val Ala Ile Gln Gly Va - #l Lys Ala Ser Leu Tyr          50              - #    55              - #    60                           - - Val Ala Met Asn Gly Glu Gly Tyr Leu Tyr Se - #r Ser Asp Val Phe Thr      65                  - #70                  - #75                  - #80        - - Pro Glu Cys Lys Phe Lys Glu Ser Val Phe Gl - #u Asn Tyr Tyr Val Ile                      85  - #                90  - #                95               - - Tyr Ser Ser Thr Leu Tyr Arg Gln Gln Glu Se - #r Gly Arg Ala Trp Phe                  100      - #           105      - #           110                  - - Leu Gly Leu Asn Lys Glu Gly Gln Ile Met Ly - #s Gly Asn Arg Val Lys              115          - #       120          - #       125                      - - Lys Thr Lys Pro Ser Ser His Phe Val Pro Ly - #s Pro Ile Glu Val Cys          130              - #   135              - #   140                          - - Met Tyr Arg Glu Pro Ser Leu His Glu Ile Gl - #y Glu Lys Gln Gly Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ser Arg Lys Ser Ser Gly Thr Pro Thr Met As - #n Gly Gly Lys Val        Val                                                                                             165  - #               170  - #               175             - - Asn Gln Asp Ser Thr                                                                  180                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 208 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Ph - #e Gly Val Gln Asp Ala      1               5   - #                10  - #                15               - - Val Pro Phe Gly Asn Val Pro Val Leu Pro Va - #l Asp Ser Pro Val Leu                  20      - #            25      - #            30                   - - Leu Ser Asp His Leu Gly Gln Ser Glu Ala Gl - #y Gly Leu Pro Arg Gly              35          - #        40          - #        45                       - - Pro Ala Val Thr Asp Leu Asp His Leu Lys Gl - #y Ile Leu Arg Arg Arg          50              - #    55              - #    60                           - - Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Gl - #u Ile Phe Pro Asn Gly      65                  - #70                  - #75                  - #80        - - Thr Ile Gln Gly Thr Arg Lys Asp His Ser Ar - #g Phe Gly Ile Leu Glu                      85  - #                90  - #                95               - - Phe Ile Ser Ile Ala Val Gly Leu Val Ser Il - #e Arg Gly Val Asp Ser                  100      - #           105      - #           110                  - - Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Gl - #u Leu Tyr Gly Ser Glu              115          - #       120          - #       125                      - - Lys Leu Thr Gln Glu Cys Val Phe Arg Glu Gl - #n Phe Glu Glu Asn Trp          130              - #   135              - #   140                          - - Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys Hi - #s Val Asp Thr Gly Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Tyr Tyr Phe Ala Leu Asn Lys Asp Gly Th - #r Pro Arg Glu Gly        Thr                                                                                             165  - #               170  - #               175             - - Arg Thr Lys Arg His Gln Lys Phe Thr His Ph - #e Leu Pro Arg Pro Val                  180      - #           185      - #           190                  - - Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys As - #p Ile Leu Ser Gln Ser              195          - #       200          - #       205                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 239 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Met Gly Leu Ile Trp Leu Leu Leu Leu Ser Le - #u Leu Glu Pro Gly Trp      1               5   - #                10  - #                15               - - Pro Ala Ala Gly Pro Gly Ala Arg Leu Arg Ar - #g Asp Ala Gly Gly Arg                  20      - #            25      - #            30                   - - Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pr - #o Arg Arg Arg Lys Leu              35          - #        40          - #        45                       - - Tyr Cys Ala Thr Lys Tyr His Leu Gln Leu Hi - #s Pro Ser Gly Arg Val          50              - #    55              - #    60                           - - Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser Il - #e Leu Glu Ile Thr Ala      65                  - #70                  - #75                  - #80        - - Val Glu Val Gly Ile Val Ala Ile Arg Gly Le - #u Phe Ser Gly Arg Tyr                      85  - #                90  - #                95               - - Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Al - #a Ser Glu His Tyr Ser                  100      - #           105      - #           110                  - - Ala Glu Cys Glu Phe Val Glu Arg Ile His Gl - #u Leu Gly Tyr Asn Thr              115          - #       120          - #       125                      - - Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Se - #r Thr Pro Gly Ala Arg          130              - #   135              - #   140                          - - Arg Gln Pro Ser Ala Glu Arg Leu Trp Tyr Va - #l Ser Val Asn Gly Lys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Ar - #g Thr Gln Lys Ser        Ser                                                                                             165  - #               170  - #               175             - - Leu Phe Leu Pro Arg Val Leu Asp His Arg As - #p His Glu Met Val Arg                  180      - #           185      - #           190                  - - Gln Leu Gln Ser Gly Leu Pro Arg Pro Pro Gl - #y Lys Gly Val Gln Pro              195          - #       200          - #       205                      - - Arg Arg Arg Arg Gln Lys Gln Ser Pro Asp As - #n Leu Glu Pro Ser His          210              - #   215              - #   220                          - - Val Gln Ala Ser Arg Leu Gly Ser Gln Leu Gl - #u Ala Ser Ala His          225                 2 - #30                 2 - #35                            - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 194 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Met His Lys Trp Ile Leu Thr Trp Ile Leu Pr - #o Thr Leu Leu Tyr Arg      1               5   - #                10  - #                15               - - Ser Cys Phe His Ile Ile Cys Leu Val Gly Th - #r Ile Ser Leu Ala Cys                  20      - #            25      - #            30                   - - Asn Asp Met Thr Pro Glu Gln Met Ala Thr As - #n Val Asn Cys Ser Ser              35          - #        40          - #        45                       - - Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Me - #t Glu Gly Gly Asp Ile          50              - #    55              - #    60                           - - Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Tr - #p Tyr Leu Arg Ile Asp      65                  - #70                  - #75                  - #80        - - Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Me - #t Lys Asn Asn Tyr Asn                      85  - #                90  - #                95               - - Ile Met Glu Ile Arg Thr Val Ala Val Gly Il - #e Val Ala Ile Lys Gly                  100      - #           105      - #           110                  - - Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Ly - #s Glu Gly Lys Leu Tyr              115          - #       120          - #       125                      - - Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Ph - #e Lys Glu Leu Ile Leu          130              - #   135              - #   140                          - - Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Ly - #s Trp Thr His Asn Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gl - #y Ile Pro Val Arg        Gly                                                                                             165  - #               170  - #               175             - - Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala Hi - #s Phe Leu Pro Met Ala                  180      - #           185      - #           190                  - - Ile Thr                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 155 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Al - #a Leu Thr Glu Lys Phe      1               5   - #                10  - #                15               - - Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Ly - #s Leu Leu Tyr Cys Ser                  20      - #            25      - #            30                   - - Asn Gly Gly His Phe Leu Arg Ile Leu Pro As - #p Gly Thr Val Asp Gly              35          - #        40          - #        45                       - - Thr Arg Asp Arg Ser Asp Gln His Ile Gln Le - #u Gln Leu Ser Ala Glu          50              - #    55              - #    60                           - - Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Gl - #u Thr Gly Gln Tyr Leu      65                  - #70                  - #75                  - #80        - - Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Se - #r Gln Thr Pro Asn Glu                      85  - #                90  - #                95               - - Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu As - #n His Tyr Asn Thr Tyr                  100      - #           105      - #           110                  - - Ile Ser Lys Lys His Ala Glu Lys Asn Trp Ph - #e Val Gly Leu Lys Lys              115          - #       120          - #       125                      - - Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr Hi - #s Tyr Gly Gln Lys Ala          130              - #   135              - #   140                          - - Ile Leu Phe Leu Pro Leu Pro Val Ser Ser As - #p                          145                 1 - #50                 1 - #55                            - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 210 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Leu Gly Asp Arg Gly Arg Gly Arg Ala Leu Pr - #o Gly Gly Arg Leu Gly      1               5   - #                10  - #                15               - - Gly Arg Gly Arg Gly Arg Ala Pro Glu Arg Va - #l Gly Gly Arg Gly Arg                  20      - #            25      - #            30                   - - Gly Arg Gly Thr Ala Ala Pro Arg Ala Ala Pr - #o Ala Ala Arg Gly Ser              35          - #        40          - #        45                       - - Arg Pro Gly Pro Ala Gly Thr Met Ala Ala Gl - #y Ser Ile Thr Thr Leu          50              - #    55              - #    60                           - - Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Al - #a Phe Pro Pro Gly His      65                  - #70                  - #75                  - #80        - - Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys As - #n Gly Gly Phe Phe Leu                      85  - #                90  - #                95               - - Arg Ile His Pro Asp Gly Arg Val Asp Gly Va - #l Arg Glu Lys Ser Asp                  100      - #           105      - #           110                  - - Pro His Ile Lys Leu Gln Leu Gln Ala Glu Gl - #u Arg Gly Val Val Ser              115          - #       120          - #       125                      - - Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Al - #a Met Lys Glu Asp Gly          130              - #   135              - #   140                          - - Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Gl - #u Cys Phe Phe Phe Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Ar - #g Ser Arg Lys Tyr        Thr                                                                                             165  - #               170  - #               175             - - Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gl - #n Tyr Lys Leu Gly Ser                  180      - #           185      - #           190                  - - Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Ph - #e Leu Pro Met Ser Ala              195          - #       200          - #       205                      - - Lys Ser                                                                      210                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 206 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Met Ser Gly Pro Gly Thr Ala Ala Val Ala Le - #u Leu Pro Ala Val Leu      1               5   - #                10  - #                15               - - Leu Ala Leu Leu Ala Pro Trp Ala Gly Arg Gl - #y Gly Ala Ala Ala Pro                  20      - #            25      - #            30                   - - Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Le - #u Glu Arg Arg Trp Glu              35          - #        40          - #        45                       - - Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pr - #o Val Ala Ala Gln Pro          50              - #    55              - #    60                           - - Lys Glu Ala Ala Val Gln Ser Gly Ala Gly As - #p Tyr Leu Leu Gly Ile      65                  - #70                  - #75                  - #80        - - Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gl - #y Ile Gly Phe His Leu                      85  - #                90  - #                95               - - Gln Ala Leu Pro Asp Gly Arg Ile Gly Gly Al - #a His Ala Asp Thr Arg                  100      - #           105      - #           110                  - - Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Ar - #g Gly Val Val Ser Ile              115          - #       120          - #       125                      - - Phe Gly Val Ala Ser Arg Phe Phe Val Ala Me - #t Ser Ser Lys Gly Lys          130              - #   135              - #   140                          - - Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cy - #s Thr Phe Lys Glu Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Se - #r Tyr Lys Tyr Pro        Gly                                                                                             165  - #               170  - #               175             - - Met Phe Ile Ala Leu Ser Lys Asn Gly Lys Th - #r Lys Lys Gly Asn Arg                  180      - #           185      - #           190                  - - Val Ser Pro Thr Met Lys Val Thr His Phe Le - #u Pro Arg Leu                      195          - #       200          - #       205                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 208 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Met Ala Leu Gly Gln Lys Leu Phe Ile Thr Me - #t Ser Arg Gly Ala Gly      1               5   - #                10  - #                15               - - Arg Leu Gln Gly Thr Leu Trp Ala Leu Val Ph - #e Leu Gly Ile Leu Val                  20      - #            25      - #            30                   - - Gly Met Val Val Pro Ser Pro Ala Gly Thr Ar - #g Ala Asn Asn Thr Leu              35          - #        40          - #        45                       - - Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu Se - #r Arg Ser Arg Ala Gly          50              - #    55              - #    60                           - - Leu Ala Gly Glu Ile Ala Gly Val Asn Trp Gl - #u Ser Gly Tyr Leu Val      65                  - #70                  - #75                  - #80        - - Gly Ile Lys Arg Gln Arg Arg Leu Tyr Cys As - #n Val Gly Ile Gly Phe                      85  - #                90  - #                95               - - His Leu Gln Val Leu Pro Asp Gly Arg Ile Se - #r Gly Thr His Glu Glu                  100      - #           105      - #           110                  - - Asn Pro Tyr Ser Leu Leu Glu Ile Ser Thr Va - #l Glu Arg Gly Val Val              115          - #       120          - #       125                      - - Ser Leu Phe Gly Val Arg Ser Ala Leu Phe Va - #l Ala Met Asn Ser Lys          130              - #   135              - #   140                          - - Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gln Gl - #u Glu Cys Lys Phe Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala Ty - #r Glu Ser Asp Leu        Tyr                                                                                             165  - #               170  - #               175             - - Gln Gly Thr Tyr Ile Ala Leu Ser Lys Tyr Gl - #y Arg Val Lys Arg Gly                  180      - #           185      - #           190                  - - Ser Lys Val Ser Pro Ile Met Thr Val Thr Hi - #s Phe Leu Pro Arg Ile              195          - #       200          - #       205                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 268 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Ph - #e Ser His Leu Ile Leu      1               5   - #                10  - #                15               - - Ser Ala Trp Ala His Gly Glu Lys Arg Leu Al - #a Pro Lys Gly Gln Pro                  20      - #            25      - #            30                   - - Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gl - #y Ser Ser Ser Arg Gln              35          - #        40          - #        45                       - - Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Al - #a Ser Ser Ser Pro Ala          50              - #    55              - #    60                           - - Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Gl - #u Gln Ser Ser Phe Gln      65                  - #70                  - #75                  - #80        - - Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Le - #u Tyr Cys Arg Val Gly                      85  - #                90  - #                95               - - Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gl - #y Lys Val Asn Gly Ser                  100      - #           105      - #           110                  - - His Glu Ala Asn Met Leu Ser Val Leu Glu Il - #e Phe Ala Val Ser Gln              115          - #       120          - #       125                      - - Gly Ile Val Gly Ile Arg Gly Val Phe Ser As - #n Lys Phe Leu Ala Met          130              - #   135              - #   140                          - - Ser Lys Lys Gly Lys Leu His Ala Ser Ala Ly - #s Phe Thr Asp Asp Cys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Ty - #r Asn Thr Tyr Ala        Ser                                                                                             165  - #               170  - #               175             - - Ala Ile His Arg Thr Glu Lys Thr Gly Arg Gl - #u Trp Tyr Val Ala Leu                  180      - #           185      - #           190                  - - Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Se - #r Pro Arg Val Lys Pro              195          - #       200          - #       205                      - - Gln His Ile Ser Thr His Phe Leu Pro Arg Ph - #e Lys Gln Ser Glu Gln          210              - #   215              - #   220                          - - Pro Glu Leu Ser Phe Thr Val Thr Val Pro Gl - #u Lys Lys Asn Pro Pro      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Al - #a Pro Arg Lys Asn        Thr                                                                                             245  - #               250  - #               255             - - Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Ph - #e Gly                                  260      - #           265                                       __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of:(a) amino acid residues 1to 255 of SEQ ID NO:2; (b) amino acid residues 2 to 255 of SEQ ID NO:2;(c) at least 30 contiguous amino acid residues of SEQ ID NO:2; (d) theamino acid sequence of the polypeptide encoded by the human cDNAcontained in ATCC Deposit No. 97150; (e) at least 30 contiguous aminoacid residues of the polypeptide encoded by the human cDNA contained inATCC Deposit No. 97150; (f) the amino acid sequence of a fragment of thepolypeptide shown as amino acid residues 1 to 255 of SEQ ID NO:2 whereinsaid fragment has fibroblast growth factor activity; and (g) the aminoacid sequence of a fragment of the polypeptide encoded by the human cDNAcontained in ATCC Deposit No. 97150 wherein said fragment has fibroblastgrowth factor activity.
 2. The isolated polypeptide of claim 1 whereinthe amino acid sequence is (c).
 3. The isolated polypeptide of claim 2wherein the polypeptide comprises amino acid residues 2 to 255 of SEQ IDNO:2.
 4. The isolated polypeptide of claim 3 wherein the polypeptidecomprises amino acid residues 1 to 255 of SEQ ID NO:2.
 5. A compositioncomprising the isolated polypeptide of claim 4 in a pharmaceuticallyacceptable carrier.
 6. An isolated polypeptide produced by the methodcomprising:(a) expressing the polypeptide of claim 4 from a host cell;and (b) recovering said polypeptide.
 7. A composition comprising theisolated polypeptide of claim 3 in a pharmaceutically acceptablecarrier.
 8. An isolated polypeptide produced by the methodcomprising:(a) expressing the polypeptide of claim 3 from a host cell;and (b) recovering said polypeptide.
 9. The isolated polypeptide ofclaim 2 wherein the polypeptide comprises at least 50 contiguous aminoacid residues of SEQ ID NO:2.
 10. A composition comprising the isolatedpolypeptide of claim 9 in a pharmaceutically acceptable carrier.
 11. Anisolated polypeptide produced by the method comprising:(a) expressingthe polypeptide of claim 9 from a host cell; and (b) recovering saidpolypeptide.
 12. A composition comprising the isolated polypeptide ofclaim 2 in a pharmaceutically acceptable carrier.
 13. The isolatedpolypeptide of claim 1 wherein the amino acid sequence is (e).
 14. Theisolated polypeptide of claim 13 wherein the polypeptide comprises theamino acid sequence of the polypeptide encoded by the human cDNAcontained in ATCC Deposit No.
 97150. 15. A composition comprising theisolated polypeptide of claim 14 in a pharmaceutically acceptablecarrier.
 16. An isolated polypeptide produced by the methodcomprising:(a) expressing the polypeptide of claim 14 from a host cell;and (b) recovering said polypeptide.
 17. A composition comprising theisolated polypeptide of claim 13 in a pharmaceutically acceptablecarrier.
 18. An isolated polypeptide produced by the methodcomprising:(a) expressing the polypeptide of claim 3 from a host cell;and (b) recovering said polypeptide.
 19. The isolated polypeptide ofclaim 1 fused to a heterologous polypeptide.
 20. A compositioncomprising the isolated polypeptide of claim 19 in a pharmaceuticallyacceptable carrier.
 21. An isolated polypeptide produced by the methodcomprising:(a) expressing the polypeptide of claim 19 from a host cell;and (b) recovering said polypeptide.
 22. The isolated polypeptide ofclaim 1 wherein the amino acid sequence is (f).
 23. A compositioncomprising the isolated polypeptide of claim 22 in a pharmaceuticallyacceptable carrier.
 24. An isolated polypeptide produced by the methodcomprising:(a) expressing the polypeptide of claim 22 from a host cell;and (b) recovering said polypeptide.
 25. The isolated polypeptide ofclaim 1 wherein the amino acid sequence is (g).
 26. A compositioncomprising the isolated polypeptide of claim 25 in a pharmaceuticallyacceptable carrier.
 27. An isolated polypeptide produced by the methodcomprising:(a) expressing the polypeptide of claim 25 from a host cell;and (b) recovering said polypeptide.
 28. A composition comprising theisolated polypeptide of claim 1 in a pharmaceutically acceptablecarrier.
 29. An isolated polypeptide produced by the methodcomprising:(a) expressing the polypeptide of claim 2 from a host cell;and (b) recovering said polypeptide.